DNA sequencing is of increasing importance in biological research, biotechnology, medical diagnostics, forensics, and other fields. Most sequencing is done by the dideoxy chain termination method (Sanger et al., 1977; Slatko et al., 1991). Sequencing reactions generally involve the dispensing of several liquid aliquots: a DNA polymerase, four deoxynucleotides, four chain terminators, and sometimes buffer or other components.
Sequencing today generally uses fluorescently labeled dideoxynucleotide terminators. The four fluorescently labeled dideoxynucleotides can be used in a single reaction mixture, and spectrally resolved with electrophoresis in a single lane. See Bergot et al., U.S. Pat. No. 5,366,860. The fluorescent labels constitute a greater chemical alteration of the nucleotides than do the radioactive labels. The fluorescent labels are complex groups that potentially can be damaged during drying. Recently, improved fluorescent labels have been introduced that are energy transfer dyes. See Lee et al., U.S. Pat. Nos. 5,800,996; 5,863,727; 5,945,526; and 6,335,440. In energy transfer dyes, a donor dye absorbs incoming radiation and transfers the excitation energy to an acceptor dye that is linked to the donor dye. The acceptor dye then emits radiation at a wavelength of lower energy than the wavelength of peak absorption for the donor dye. Energy transfer dyes offer improved fluorescence yield and better spectral resolution of the four dyes needed in a sequencing reaction. However, they are larger and more complicated molecules than standard dyes, and thus could be more sensitive to damage by desiccation or other environmental stress.
There is a need for methods of DNA sequencing, and other methods of nucleic acid analysis involving nucleic acid labeling, that use dried fluorescent reagents. There is also a need for methods in which the polymerase and all reagents, including four labeled chain terminators, are dried in a single well to eliminate the need to transfer solutions between wells.